Structural remodeling of gray matter astrocytes in the neonatal pig brain after hypoxia/ischemia

Abstract

Astrocytes play a vital role in the brain; their structural integrity and sustained function are essential for neuronal viability, especially after injury or insult. In this study, we have examined the response of astrocytes to hypoxia/ischemia (H/I), employing multiple methods (immunohistochemistry, iontophoretic cell injection, Golgi-Kopsch staining, and D-aspartate uptake) in a neonatal pig model of H/I. We have identified morphological changes in cortical gray matter astrocytes in response to H/I. Initial astrocytic changes were evident as early as 8 h post-insult, before histological evidence for neuronal damage. By 72 h post-insult, astrocytes exhibited significantly fewer processes that were shorter, thicker, and had abnormal terminal swellings, compared with astrocytes from control brains that exhibited a complex structure with multiple fine branching processes. Quantification and image analysis of astrocytes at 72 h post-insult revealed significant decreases in the average astrocyte size, from 686 microm(2) in controls to 401 microm(2) in H/I brains. Sholl analysis revealed a significant decrease (>60%) in the complexity of astrocyte branching between 5 and 20 microm from the cell body. D-Aspartate uptake studies revealed that the H/I insult resulted in impaired astrocyte function, with significantly reduced clearance of the glutamate analog, D-aspartate. These results suggest that astrocytes may be involved in the pathophysiological events of H/I brain damage at a far earlier time point than first thought. Developing therapies that prevent or reverse these astrocytic changes may potentially improve neuronal survival and thus might be a useful strategy to minimize brain damage after an H/I insult.

Phosphorylation of GFAP is Associated with Injury in the Neonatal Pig Hypoxic-Ischemic Brain

“…We have demonstrated pre-gliotic astrocyte changes are evident from 1-8h after an HI insult, prior to histological evidence of neuronal damage [6,29]. At 72h post-insult, overall astrocyte size decreased by 30-40% in grey and white matter and these astrocytic changes were associated with histological brain damage [6,28]. Our results and others confirm the importance of neuron-glia interactions in normal brain function [30].…”

“…This theory is supported by Martin et al [23] who demonstrated a significant increase in GFAP immunoreactivity in the HI pig brain at 96h post-insult. Our previous studies have demonstrated that the post-HI brain undergoes significant changes in the expression of various astrocytic and neuronal proteins and the structure of grey and white matter astrocytes [6,[24][25][26][27][28][29]. We have demonstrated pre-gliotic astrocyte changes are evident from 1-8h after an HI insult, prior to histological evidence of neuronal damage [6,29].…”

“…The few astrocytes that expressed pGFAP in long processes had atypical features such as thickened, varicose processes (Fig. 4D) and did not exhibit the fine, bushy processes of a normal control astrocyte (see images contained within [6,39]). …”

“…Our previous studies have shown that GFAP (via NHERF1 and ezrin) anchors the glutamate transporter GLAST in the plasma membrane, enhancing glutamate transport [6].…”

“…Our previous studies have demonstrated that the post-HI brain undergoes significant changes in the expression of various astrocytic and neuronal proteins and the structure of grey and white matter astrocytes [6,[24][25][26][27][28][29]. We have demonstrated pre-gliotic astrocyte changes are evident from 1-8h after an HI insult, prior to histological evidence of neuronal damage [6,29]. At 72h post-insult, overall astrocyte size decreased by 30-40% in grey and white matter and these astrocytic changes were associated with histological brain damage [6,28].…”